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. 2020 Dec 21;15(12):e0244158. doi: 10.1371/journal.pone.0244158

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© 2020 Lin et al

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Fig 1 — (A) Flowchart with timeline to isolate IgG⁺/Ab1 Fab⁺/ Ab1^ctrl Fab^- B cells from Ab1 Fab immunized rabbit peripheral blood for culture, primary ELISA screening, molecular cloning and recombinant IgGs expression to identify Ab1 CDRs-specific Ab2 for characterization (B) IgG⁺ B cells sorted out from immunized rabbit peripheral blood were further enriched for Ab1 Fab⁺/Ab1^ctrl Fab^-/IgG⁺ B cells by Flow Cytometry (C) High serum titer of 3 rabbits against Ab1 Fab by ELISA (vs. pre-immunized serum control) demonstrated robust Ab1 immunization (D) High-throughput primary ELISA screening using cultured rabbit B cell supernatants confirmed majorities of clones were Ab1 Fab specific (OD > 0.25), but not Ab1^ctrl Fab (OD < 0.1) (E) Purified recombinant IgGs of 24 unique clones were reconfirmed specificity against Ab1 Fab but not to Ab1^ctrl Fab and other human control Fab (Hu^ctrl Fab) derived from native IgGs in normal human plasma. Ab1, antibody therapeutics; Ab2, anti-IDs.